mmp-1 is a (pre-)invasive factor in barrett-associated ... · mmp-1 is a fibroblast-type or...

RESEARCH Open Access

MMP-1 is a (pre-)invasive factor in Barrett-associated esophageal adenocarcinomas and isassociated with positive lymph node statusMartin Grimm1†, Maria Lazariotou2†, Stefan Kircher3, Luisa Stuermer1, Christoph Reiber1, Andreas Höfelmayr1,Stefan Gattenlöhner4, Christoph Otto1, Christoph T Germer1, Burkhard HA von Rahden1*

Abstract

Background: Esophageal adenocarcinomas (EACs) arise due to gastroesophageal reflux, with Barrett’s esophagus(BE) regarded as precancerous lesion. Matrix metalloproteinases (MMPs) might play a role during the multistepcarcinogenetic process.

Methods: Expression of MMP-1 and -13 was analyzed in esophageal cancer (n = 41 EAC with BE, n = 19 EACwithout BE, and n = 10 esophageal squamous-cell carcinomas, ESCC), furthermore in BE without intraepithelialneoplasia (IN) (n = 18), and the cell line OE-33. MMP-1 was co-labelled with Ki-67 (proliferation), Cdx-2 (marker forintestinal metaplasia, BE) and analyzed on mRNA level. MMP-1 staining results were correlated withclinicopatholocical parameters.

Results: On protein level, MMP-1 expression was found in 39 of 41 (95%) EAC with BE, in 19 of 19 (100%) EACwithout BE, in 6 of 10 (60%) ESCC, and in 10 of 18 (56%) BE without IN. No expression of MMP-13 was found inthese specimens. Quantification showed 48% MMP-1 positive cells in EAC with BE, compared to 35% in adjacentBE (p < 0.05), 44% in EAC without BE, 32% in ESCC, and 4% in BE without IN. Immunofluorescence double stainingexperiments revealed increased MMP-1 expressing in proliferating cells (MMP-1+/Ki-67+) (r = 0.943 for BE and r =0.811 for EAC). On mRNA-level, expression of MMP-1 was significantly higher in EAC compared to BE (p = 0.01) andconfirmed immunohistochemical staining results. High MMP-1 levels were associated with lymph node metastasesbut not with poorer survival (p = 0.307).

Conclusions: Our findings suggest that MMP-1 plays a role as preinvasive factor in BE-associated EAC. Expressionof MMP-1 in proliferating BE and EAC cells suggest malignant proliferation following the clonal expansion model.

BackgroundEsophageal Adenocarcinomas and Barrett’s EsophagusEsophageal adenocarcinoma is an entity of increasingclinical importance, due to an unexplained incidencerise among white males in the Western world [1], and adismal prognosis [2,3]. Chances for cure are still limitedto early, surgically resectable tumor stages, prior to sys-temic dissemination of the disease. Esophageal adeno-carcinomas develop almost exclusively in the distal third

of the esophagus, under the chronically damaging effectof gastroesophageal reflux [2,3]. Barrett’s esophagus -defined as columnar lined epithelium in the distal eso-phagus, characterized by specialized intestinal mucosa(with goblet cells) - is regarded as precancerous lesion,giving rise to these tumors.Malignant progression within Barrett’s esophagus (BE)

is regarded to follow a sequence of well-characterizedhistopathologic changes, from intestinal metaplasia, overlow-grade and high-grade intraepithelial neoplasiatowards invasive esophageal adenocarcinomas (EAC)[2,3]. However, not all EACs are associated with BE insurgical series [4,5], and only a minority of patients with

*Correspondence: [email protected]†Contributed equally1Department of General-, Visceral-, Vascular and Pediatric Surgery, Universityof Wuerzburg Hospital, Oberduerrbacher Strasse 6, 97080 Wuerzburg,GermanyFull list of author information is available at the end of the article

Grimm et al. Journal of Translational Medicine 2010, 8:99http://www.translational-medicine.com/content/8/1/99

© 2010 Grimm et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

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Barrett’s esophagus finally progress to cancer, with anincidence between 0.5 and 2.0% per year [6].These and other findings have raised doubt about the

relevance of Barrett’s esophagus as the precancerouslesion of EAC (e.g. [7]), stimulating the search for thecell population, from which esophageal adenocarcino-mas originate, which is currently unknown.The cell that gives rise to Barrett metaplasia is not

known. Recently, it has been hypothesized that intestinalmetaplasia may arise from a change in the differentia-tion pattern of stem cells that either reside in the eso-phagus or are recruited via the hematogenous routefrom the bone marrow [8]. In addition, due to the mul-tistep carcinogenesis, the clonal selection model impliesthat malignant transformation occurs by multiple muta-tions in a random single cell and subsequent clonalselection takes place [9-11].Evidence is accumulating, that matrix metalloprotei-

nases (MMPs) may drive carcinogenesis according to amodel of multistep carcinogenesis or a cancer stem cellhypothesis mediated by the integrin collagen receptoralpha(2)beta(1)-integrin pathway, which may also applyto esophageal adenocarcinomas [11-15]. MMPs are afamily of highly homologous protein-degrading zincdependent enzymes, functioning as endopeptidases. Thisfamily currently includes more than 25 members thatcan be divided into collagenases (MMP-1, -8, and -13),gelatinases (MMP-2 and 9), stromelysins (MMP-3 and10), matrilysins (MMP-7 and 26), and the membrane-type MMPs (MMP-14 to 17 and 24). FurthermoreMMPs are able to degrade the basement membrane ofvessels which is essential for tumor invasion into bloodand lymph vessels [14,16,17].MMP-1 is a fibroblast-type or interstitial collagenase

and majorly secreted from fibroblasts, keratinocytes,macrophages, but also cancer cells. MMP-13 is anothertumor-derived MMP that is implicated to have coopera-tive effects with MMP-1 and is related to cancer aggres-siveness [18]. No data are currently available whichconnect expression of MMP-1 and MMP-13 withBarrett’s metaplasia and related EACs with the tumorproliferation model of multistep carcinogenesis and clini-copathologic features. The aim of our study was to inves-tigate expression of collagenases MMP-1 and -13 in EAC(with and without associated BE) as well as non-dysplas-tic BE (without evidence of intraepithelial neoplasia andcarcinoma) and ESCC. We aimed to indicate their poten-tial role as preinvasive factors in BE, to compare expres-sion levels with adjacent EACs, and to investigate apotential impact of MMP expression on survival, as wellas correlation with clinicopathologic features.

MethodsPatients and Tumor SpecimenSurgical specimen from altogether 70 patients, havingundergone primary surgical resection for esophagealcancer between January 2001 and June 2004 wereincluded in our study, furthermore n = 18 biopsies frompatients with non-dysplastic BE (without evidence ofinvasive carcinoma or intraepithelial neoplasia). Patientshaving undergone preoperative antineoplastic therapies(chemoradiation/chemotherapy) were excluded. Onlypatients in whom complete (R0) resection had beenachieved were included.We used archieval formalin-fixed, paraffin-embedded

tissue from routine histopathologic work-up, which hadbeen performed under standardized conditions. Thematerial had been stored with permission of the localethics committee, after informed consent obtained fromthe patients prior to surgical resection.There were n = 41 esophageal adenocarcinomas

(EAC) with associated Barrett’s esophagus (BE), n = 19EAC without BE and n = 10 esophageal squamous-cellcarcinomas of the esophagus (which were intended toserve as positive control for MMP-1 expression), and n =18 Barrett’s biopsies without intraepithelial neoplasia orinvasive carcinoma which were derived from patientswith gastroesophageal reflux disease (GERD). EACwithout BE was defined based on clinical information(endoscopic evidence of Barrett’s mucosa), throughwork-up of all tumor blocks (searching for specializedintestinal metaplasia) and Cdx-2 staining performed inaddition, which has a 70% sensitivity for stainingintestinal metapasia [19].Of note, the 19 EAC without BE were tumors in the

distal esophagus (AEG type I tumors, according to theclassification by Siewert and Stein, 1998, Br J Surg [20]),and explicitly not localized at the level of the anatomicgastric cardia (AEG type II tumors). The AEG type IIadenocarcinoma is a tumor entity on its own and mustbe discussed differently.Follow-up data were obtained from our local tumor

registry of Lower Franconia/Germany. This tumor regis-try documents all cancer patients in the area of LowerFranconia/Germany. Information were obtained accord-ing to clinical visits of the patients (after 6 months, 12months, 18 months, and thereafter one clinical visit peryear). Information about patients who did not partici-pate in follow-up investigations were obtained from gen-eral practitioners. Follow-up was complete for allpatients (100%). Mean follow-up accounted for (29months ± 17.6 standard deviation). Patient and tumorcharacteristics are given in Table 1.


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Histopathologic Analysis, Tumor Staging and Definition ofBarrett’s mucosaTumor blocks of paraffin-embedded tissue were selectedby two experienced gastrointestinal pathologists (StefanKircher, Stefan Gattenlöhner) on routine hematoxylinand eosin (H&E) stained sections. Sections from allavailable tumor blocks of all cases underwent intensivehistopathologic assessment, blinded to the prior histo-pathology report. H&E stained sections were analyzedwith special focus on tumor infiltrated areas (EAC/ESCC), stromal areas and infiltrating immune cells.Tumor staging was performed according to the 6th edi-tion of the TNM staging system by the UICC/AJCC of2002 [21]. Grading was performed according to WHOcriteria [22].

Tumor characteristics (UICC stage, pT-categories, pN-categories, cM-categories, number of removed lymphnodes, number of tumor infiltrated lymph nodes, resi-dual tumor status, location) and patient characteristicswere documented in a database (EXCEL, Microsoft).Barrett’s mucosa was defined as specialized intestinal

metaplasia (IM), characterized by goblet cells anddisturbed glandular architecture [2,3]. In addition,immunohistochemistry with caudal type homeoboxtranscription factor 2 (Cdx-2), which is suggested asearly marker for intestinal metaplasia [23], was used toidentify tiny foci of intestinal metaplasia.Furthermore, different degrees of high-grade and low-

grade intraepithelial neoplasia within Barrett’s mucosawere assessed. EACs were classified as “EAC with BE”,when at least tiny foci of intestinal metaplasia werefound due to Cdx-2 staining. EAC were classified as“EAC without BE”, when the pathologists could not findintestinal metaplasia on any of the tumor blocks.

Immunohistochemical and immunofluorescent stainingUnconjugated MMP-1, Ki-67, and isotype control anti-bodies were purchased from Acris (Hiddenhausen,Germany). The unconjugated Cdx-2 antibody wasobtained from Biogenex (San Ramon, USA) and theunconjugated MMP-13 was provided by NeoMarkers(Asbach, Germany). The secondary antibody used forimmunofluorescence double staining of Ki-67 was afluoresceinisothiocyanat (FITC)-conjugated AffiniPuredonkey-anti-rabbit IgG, used at 1:200 dilution (JacksonImmunoResearch Laboratories Inc., Suffolk, England).The secondary antibody for MMP-1 was a Cy3-conjugatedAffiniPure donkey-anti-mouse IgG (Jackson Immuno-Research), used at 1:200 dilution.

Cell CultureWe analyzed MMP-1 and MMP-13 expression in cells(1 × 104) from the esophageal adenocarcinoma cell lineOE-33 (Sigma-Aldrich, Steinheim, Germany) in cytos-pins. This cell line is the only commercially availableadenocarcinoma cell line of the lower esophagus (Bar-rett’s metaplasia) and was established from a 73-year-oldfemale patient. The tumor was identified as pathologicalstage IIA (UICC) and showed poor differentiation.Using RT-PCR we tested negative for mycoplasma con-tamination of this cell line that was provided to ourlaboratory in December 2009 by Sigma. The cell linewas cultured in RPMI-1640 medium, supplemented with10% Fetal Bovine Serum, 100 units/ml of penicillin and100 μg/ml of streptomycin. Cytospins of the OE-33 cellline were fixed in acetone and dried for 10 minutes.Rehydration, blocking, and the staining procedure stepswere the same as described for immunohistochemistry

Table 1 Clinicopathological characteristics of the EAC studypopulation (with and without histological proven BE)

Characteristics Patients(n = 60)

MMP-1 expressionEAC

p-value

Low(<46%)

High(≥ 46%)

Age (y) .605

<66 30 (50%) 13 (43%) 17 (57%)

≥66 30 (50%) 14 (47%) 16 (53%)

Gender .465

Male 52 (87%) 24 (46%) 28 (54%)

Female 8 (13%) 5 (63%) 3 (37%)

Histologicalclassification

.032†

G1 17 (28%) 11 (65%) 6 (35%)

G2 22 (37%) 12 (55%) 10 (45%)

G3/4 21 (33%) 6 (29%) 15 (71%)

Depth of invasion .163††

pT1 16 (27%) 9 (56%) 7 (44%)

pT2 26 (43%) 14 (54%) 12 (46%)

pT3 10 (17%) 3 (30%) 7 (70%)

pT4 8 (13%) 3 (38%) 5 (62%)

Lymph nodesmetastasis

.016

pN0 23 (38%) 16 (70%) 7 (30%)

pN1-3 37 (62%) 13 (35%) 24 (65%)

UICC stage .163†††

UICC I 14 (23%) 8 (57%) 6 (43%)

UICC II 28 (47%) 15 (54%) 13 (46%)

UICC III 18 (30%) 6 (33%) 12 (67%)

UICC IV 0 (0%) 0 (0%) 0 (0%)

Median OS (m) 43 m 47(n = 29)

38 (n = 31)

EAC, esophageal adenocarcinomas; BE, Barrett metaplasia; y, years; G, grading;UICC, International Union against Cancer; R, residual tumor; OS, overallsurvival; m, months.†G1/2 vs. GT3/4; ††pT1/2 vs. pT3/4; †††UICC I/II vs. UICC III/IV.


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of FFPE sections. Additionally, RT-PCR was performedfor MMP-1 gene expression of OE-33 cells.

Double Staining Experiments (IF and IHC)The sequential immunofluorescence (IF) double staining(co-expression) was analyzed for MMP-1 with Ki-67expression. Sequential immunohistochemical (IHC) dou-ble staining was performed for Cdx-2 and MMP-1.

Processing of tissue and staining procedureFirst we assessed H&E sections from each tumor tissueto differentiate between BE, tumor cell areas, stromalareas and infiltrating immune cells. We then stained forMMP-1, -13, Cdx-2, and Ki-67 in additional serial sec-tions of 2 μm thickness. Tissue sections (2 μm thick-ness) were cut from paraffin blocks on a microtome andmounted on adhesive microscope slides (Hartenstein,Wuerzburg, Germany). Serial sections were deparaffi-nized in xylene and ethanol and rehydrated in water.Heat induced epitope retrieval (HIER) was performedwith citrate buffer pH 6.0 (Dako, Hamburg, Germany).For IF, slides were then incubated in normal serum (2%)and bovine serum albumin (BSA) (0.5%) at room tem-perature for 20 minutes to block nonspecific binding.Subsequently, slides were incubated with the primaryantibody or control antibody overnight at 4°C ina humidified chamber and with secondary FITC-conjugated antibody for 30 minutes at room tempera-ture in a humidified chamber. The slides were incubatedwith the second primary antibody diluted in TBS plus0.5% BSA overnight at 4°C, followed by incubation withthe secondary Cy3-conjugated antibody for 30 minutesat room temperature. Slides were counterstained withDAPI (4’,6-Diamidino-2-phenylindoldihydrochlorid,Sigma-Aldrich) and covered with Polyvinyl-alcoholmounting medium (DABCO, Sigma-Aldrich) and ana-lyzed using a Zeiss camera (Jena, Germany). The photo-graphed images using the Metamorph software package(Visitron Systems, Puchheim, Germany) were importedinto the Microsoft Office Picture Manager.For IHC, the pretreatment procedure (fixation, depar-

affinization, rehydration, HIER, and blocking) of theslides was the same as described for IF. For immuno-histochemical analysis a four-step immunoperoxidaselabeling for single antigens in formalin-fixed, paraffin-embedded sections was used as described [24]. Forimmunohistochemical double staining, we first used analkaline phosphatase (AP)-conjugated AffiniPure Donkeyanti-mouse Ab followed by 20 minutes of incubationwith Fast Red (Dako). After incubation with the secondprimary antibody, we used a horseradish peroxidase(HRP)-conjugated AffiniPure Donkey anti-rabbit IgG(Jackson ImmunoResearch) followed by 5 minutes ofincubation with DAB (Biogenex).

Quantification of Immunohistochemistry (IHC) andImmunofluorescence (IF)MMP-1 and Ki-67 IHC was quantified in EAC with BE,as well as in the associated Barrett’s mucosa, as well asEAC without BE. Quantification of immunoenzymaticstaining of IN or tumor cells was performed, analyzingsix representative individual high power fields (×400) foreach sample. Scoring was done by means of cell count-ing. The results were expressed as percentages (numberof positive cells within 100 counted tumor cells, %). Sec-tions were evaluated by two independent blinded inves-tigators separately. In case of discrepancies, bothevaluated the slides simultaneously and made an agree-ment. For each tumor section, quantification of IF dou-ble staining was performed by counting Ki-67+ cells insix microscopic high power fields (400 × magnification)in parallel with MMP-1+. The proportion of Ki-67 posi-tivity in counted MMP-1+ cells was expressed inpercentages.

Real-time quantitative reverse transcription-PCR analysisTo analyze gene expression of MMP-1 by RT-PCR inFFPE tissue, we extracted total cellular RNA and per-formed cDNA synthesis using the Absolutely RNA FFPEKit and the AffinityScript QPCR cDNA Synthesis Kitfrom Stratagene (Waldbronn, Germany). Areas of inter-est for each tissue section were manually microdissected.For both groups (BE and EAC) equal amounts of tissueareas were assessed (2 × 1.5 cm2 surface area per section,thickness of 10 μm). For OE-33 cell line, after homogeni-zation Diethyl pyrocarbonate (DEPC)-75% ethanol wasadded to the lysate to provide ideal binding conditions.The lysate was then loaded onto the RNeasy silica mem-brane (“RNeasy Mini spin column”, RNeasy Mini Kit Qia-gen, Hilden, Germany). RNA binds, and all contaminantswere washed away efficiently. Pure, concentrated RNAwas eluted in water and stored at -70°C until further analy-sis. The amount of total RNA was determined by measur-ing absorbance at 260 nm. The purity of total RNA wasestablished by confirming that the 260 nm:280 nm ratiowas within a 1.8-2.0 range, indicating that the RNA pre-parations were free of protein contaminants. Primerswere designed using the Primer Express software for pri-mer design to amplify short segments of 50-150 basepairs of target cDNA. The MMP-1 forward primersequence was: 5′-TGCTGCTGCTGCTGTTCTGGG-3′;the MMP-1 reverse primer sequence was: 5′-GGCCG-ATGGGCTGGACAGGA-3′. Matched human esophagealcDNA was purchased by BioChain (Hayward, CA, USA)as control. The housekeeping gene Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for relativequantification and cDNA quality control. The GAPDHforward primer sequence was: 5′-ATCCCATCACCA-TCTTCCAGG-3′; the GAPDH reverse primer sequence


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was: 5′-CGCCCCACTTGATTTTGG-3′. RT-PCR reac-tions were carried out as described previously [25]. Therelative quantification value, fold difference, is expressedas 2-ΔΔCt.

Statistical analysisStatistical analysis was performed with MedCalc Soft-ware, Version 11.3.2 (Mariakerke, Belgium). All valueswere expressed as Median ± Interquartile Range (IQR)because D’Agostino-Pearson test did not show a normaldistribution of gene and protein expression. Therefore,the Median value was chosen to divide patients in twodifferent groups. Survival time was determined as thetime from tumor resection to tumor conditional deathand as the time from tumor resection to time of obviousrecurrence. The overall survival (OS) time in associationwith MMP-1 expression was estimated using theKaplan-Meier method [26]. To analyze differences inthe overall/tumor related survival among patients aftersuccessful (R0) curative surgical resection for EACpatients were divided into two subgroups (dichotomousvariables). Median cut-off value for either high or lowexpressors was set at 46% for MMP-1 expression in allEAC (n = 60). The log rank test was used to check forstatistical differences between the survival curves. Caseswith less than 10% positive cells were regarded asnegative.Multivariate analyses were performed using the Cox

Proportional Hazards Model. All parameters that werefound significant on univariate analysis were included [27].Correlation analysis was performed by the non-para-

metric Spearman Rho rank correlation coefficient. Fish-er’s exact test was used to investigate the relationbetween two categorical variables. Data were analyzedusing the non-parametric Mann-Whitney U test orKruskal-Wallis test when more than 2 groups werecompared. P values of less than 0.05 were regarded sta-tistically significant.

ResultsMMP-1 expression is associated with BE and associatedEACMMP-1, MMP-13 and Cdx-2 were not expressed innormal esophageal squamous epithelium. MMP-1expression in stromal cells was considerably weak andstrongly associated with high-grade and low-gradeintraepithelial neoplasia within Barrett’s mucosa aswell as cancer cells. 95% (n = 39/41) of the patientswith BE and adjacent EAC expressed MMP-1 withinthe tumor. Similarly, 100% (n = 19/19) of the EACwithout BE expressed MMP-1, 6 of 10 ESCC (60%),and 10 of 18 (56%) Barrett’s biopsies without intrae-pithelial neoplasia or carcinoma stained positive forMMP-1.

Expression of MMP-13 was negative in tumor speci-men and in the esophageal adenocarcinoma cell lineOE-33, but was rarely detected in stromal cells (data notshown).Furthermore, we analyzed positivity of all counted

cells according to the precursor lesion and tumor entity.Compared to BE (GERD) without intraepithelial neopla-sia or carcinoma (Figure 1a, Table 2), MMP-1 expres-sion was significantly upregulated in BE (Figure 1a,Table 2) with adjacent EAC (Figure 1a, Table 2) andEAC without BE (Figure 1a, Table 2). No differences ofMMP-1 expression were found between differentdegrees in high-grade and low-grade intraepithelial neo-plasia within Barrett’s mucosa. Median MMP-1 expres-sion of all EACs (n = 60) was 46%, IQR 39.0 - 55.5%;95% CI 43.0 - 54.0%.ESCC showed significantly decreased MMP-1 expres-

sion (Figure 1a, Table 2), compared to EACs. Foradenocarcinomas without BE, the results of MMP-1expression were comparable with the higher expressionlevels of adenocarcinomas from BE. Expression levels ofMMP-1 in ESCC did not differ significantly from BEwith adjacent EAC but showed a decrease compared toBE (Figure 1a, Table 2).Figure 1b demonstrates a representative example of

MMP-1 expression in early BE. We confirmed areasanalyzed for MMP-1 expression of BE by immunohisto-chemical double staining with Cdx-2 (Figure 1c). Figure1d demonstrates a representative example of MMP-1expression in EAC. Stainings from the OE-33 adenocar-cinoma cancer cell line in cytospins served as additionalpositive control for MMP-1 expression and showed 65%positive cells (Figure 2a).

Analysis of MMP-1 gene expressionTo confirm the results of the immunohistochemicalstaining, gene expression of MMP-1 in human BE andEAC were assessed. MMP-1 gene expression in BE(Median 3.6-fold difference compared to normal tissue;IQR 3.275 to 4.625-fold difference; n = 5) was signifi-cantly (p = 0.01) lower in comparison to EAC withoutBE (Median 7.9-fold difference compared to normal tis-sue; IQR 6.3 to 8.95-fold difference; n = 4; Figure 2b).These results confirmed increased MMP-1 expression inBE and significantly elevated expression of MMP-1 inEAC without BE as observed by immunohistochemistry.RT-PCR results for MMP-1 expression in the adenocar-cinoma cell line OE-33 showed a 4.1-fold higher expres-sion compared to normal tissue.

MMP-1 expression is strongly correlated withproliferating (Ki-67+) Barrett and EAC cellsFor investigation of proliferating cells in BE and EACand its relation to multi-step carcinogenesis, we


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analyzed MMP-1 expression in early Barrett cells,adjacent EAC, EAC without BE and ESCC. Evaluationof immunohistochemically stained serial sectionsshowed a strong positive correlation of MMP-1expression with proliferating cells (Figure 3a and 3b:MMP-1+/Ki-67+: r = 0.943 for BE, n = 41 and r =0.811 for EAC, n = 60). As shown in Figure 3c by animmunofluorescence double staining, MMP-1 was co-expressed with great amounts of proliferating (Ki-67+)

cells in areas which were associated with early BE(goblet cells as well as Cdx-2 positivity wereobserved in serial sections) (Figure 3c, representativeexample of n = 41 BE). IF double staining confirmedcorrelation analysis evaluated in IHC serial sections.We found a dominant population of proliferatingMMP-1+/Ki-67+ cells in BE and EAC. Proliferationstatus (Ki-67+) itself did not have had any impact onsurvival (data not shown).

Figure 1 Immunohistochemical analysis and staining of MMP-1 in human BE and EAC. In comparison to BE without intraepithelial neoplasia(GERD) (1) a significantly (p < 0.05) increased expression of MMP-1 was observed in BE adjacent to EACs (2). Expression levels of MMP-1 weresignificantly (p < 0.05) increased in associated EACs (3) and EACs without BE (4). ESCC showed significantly (p < 0.05) decreased MMP-1 expressioncompared to EACs (5). Analysis refers to percentages of positivity of all counted cells. Grey lines show 95% confidence intervals. Statisticallysignificant values from BE and ESCC to EACs are indicated with asterisks (a). Increased expression of MMP-1 (b) was observed in early BE (arrows).Adjacent normal tissue stained negative for MMP-1 (asterisk). Single staining of MMP-1 in BE was confirmed by immunohistochemical doublestaining (c), showing Cdx-2 (nuclear staining pattern, Fast red) and MMP-1 (cytoplasmic staining pattern, brown). Significantly increased MMP-1expression was observed in adenocarcinomas compared to BE (d). Original magnification: top × 100, bottom × 200.


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Prognostic value of MMP-1 in adenocarcinomasTo analyze survival differences of patients after success-ful (R0) curative surgical resection for EAC with andwithout BE, patients were divided into two subgroups asdescribed above (dichotomous variables). Lymph nodemetastasis (pN+, Table 3, pT-category (pT3/4, Table 3)and grading (G3/4, Table 3) were shown to be unfavor-able factors in univariate analysis of all (n = 60) EACs.Moreover, we found a strong association between highMMP-1 expression and positive lymph node metastases(p = 0.016136582, Fisher’s exact test, Table 1) in EACpatients (n = 60). To analyze differences in tumorrelated survival dependent on MMP-1-expression inEAC we divided the patients in two subgroups asdescribed above (dichotomous variables). Survival insubgroup with high MMP-1 expression of all EACs (n =60; Figure 4, Table 1) was not significantly worse incomparison to the subgroup of patients with low expres-sion of MMP-1 (Figure 4, Table 1). Data show thatMMP-1 expression in BE and adjacent EACs is asso-ciated with clinicopathologic features which may predictworse clinical outcome of adjacent EACs. Multivariateanalysis using the Cox Proportional Hazards Modeldemonstrate lymph node metastases and grading as

independent prognostic factors in all (n = 60) EACs(Table 4).

Discussion‘Tissue invasion and metastasis’ is one of the six ‘hall-marks of cancer’, initially described by Hanahan andWeinberg [28,29]. Invasive and metastatic capabilitiesare largely mediated by extracellular proteases, whichare able to destroy the environment of the tumor cell.MMPs have been shown to be involved in this process.The early work of Liotta et al. [30] provided some ofthe first evidence that MMPs were involved in invasionof a tumor cell through the basement membrane. Pre-vious studies described MMP-9 in the pathogenesis ofBarrett’s esophagus, ESCC, and gastric cancer [31-33].However, we have focused on MMP-1 and MMP-13expression, because no data have been available regard-ing clinicopathological factors in BE and EAC so far.Our findings of an increased MMP-1 expression in EACis well in line with results obtained in other cancer enti-ties and few samples of EAC without clinicopathologicalassociation [34-41] suggesting a putative role in inva-sion, metastasis and poorer survival. In this context,MMP-13 has been shown to play a role in tumor

Table 2 MMP-1 expression of the study population in different tissues

Tissue n Median expression (%) IQR (%) 95% CI p-value

BE without intraepithelial neoplasia or carcinoma (GERD) 18 4 0-11 0-10.603

BE adjacent EAC 41 35 23.0-41.5 31.284-39.0 <0.05†

Adjacent EAC to BE 41 48 39.0-56.5 43.0-54.239 <0.05††

EAC without BE 19 44 39.0-55.8 39.0-55.218 <0.05††

ESCC 10 27 0-50.2 0-29.2 <0.05†††

BE, Barrett metaplasia; GERD, Gastro-Esophageal Reflux Disease; EAC, esophageal adenocarcinomas; ESCC, esophageal squamous-cell carcinomas; †significance isrelated to GERD; ††significance is related to BE with adjacent EAC; †††significance is related to EACs.

Figure 2 Immunohistochemical staining of MMP-1 from the OE-33 adenocarcinoma cancer cell line and MMP-1 gene expression inhuman BE and EAC. MMP-1 staining in cytospins from the OE-33 adenocarcinoma cancer cell line served as additional positive control (left)and showed 65% positive cells; IgG control (right) (a). Gene expression of MMP-1 in human BE and EAC. MMP-1 gene expression in BE wassignificantly (p = 0.01) lower in comparison to EAC without BE. Normal tissue is considered as one-fold (b). Statistically significant value isindicated with an asterisk.


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Figure 3 Correlation and co-expression of Ki-67 with MMP-1. Correlation from quantified immunohistochemical staining results of MMP-1+in BE (n = 41) and EAC (n = 60) with proliferating cells (Ki-67+) showed that Ki-67+ expression in BE (a) and EACs (b) had a strong directcorrelation with the expression of MMP-1+ (r = 0.943 for BE and r = 0.811 for EAC). (c) Images demonstrate a representative example of Ki-67co-expression with MMP-1+ by an immunofluorescent double staining in early BE showing the majority of proliferating (Ki-67+) cells with MMP-1+(big arrows). Small arrows indicate goblet cells. FITC green Fluoresceinisothiocyanat, Cy3 red, and DAPI 4’,6-Diamidino-2-phenylindoldihydrochloridblue. Top, Calibration bar represents 50 μm. Bottom, calibration bar represents 25 μm. The square box at the bottom demonstrates the area whichis also shown in larger magnification.


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progression of other gastrointestinal tumor entities suchas colorectal carcinoma [42], and hepatocellular carci-noma [43]. In gastric cancer and specimen with humanesophageal carcinomas (ESCC) MMP-13 expression hasbeen previously shown to contribute to malignant pro-gression. It has furthermore been suggested that itscoordinate overexpression with MMP-1 and/or MMP-2may have a synergetic effect in tumor progression[18,44]. In sharp contrast to these findings, in our study,MMP-13 was not found expressed in EACs, but wasoccasionally detected in surrounding stromal cells.Therefore, based on our results, we may exclude thiscooperative effect in patients with EAC.Our findings of high MMP-1 expression being asso-

ciated with lymph node metastases in patients with EACindicate that MMP-1 expression may be involved in

promotion of cancer progression in addition to otherclinicopathological characteristics. However, we havenot been able to demonstrate a negative impact ofincreased MMP-1 expression on survival.To date molecular pathogenesis of BE is poorly under-

stood. According to the clonal evolution model wefound a dominant population of proliferating cells (Ki-67+) in EAC, which may drive multi-step carcinogenesis[45]. We have chosen to study proliferation with Ki-67,because it is a proliferation-associated nuclear antigenand expressed in all cycling cells except for resting cellsin the G0 phase, which implies no negative survivaleffect [46] but may be associated with neoplastic pro-gression in BE [47]. Therefore, our results may indicatethat MMP-1 expression is associated with multistep car-cinogenesis according to clonal selection model from BEto EAC. Furthermore, we hypothesize MMP-1 signalingin the pathogenesis of adenocarcinomas by the integrincollagen receptor alpha(2)beta(1)-integrin pathwaywhich has been shown to be involved human prostateepithelial stem cells [15]. In osteosarcoma cells the levelof cell surface alpha(2)beta(1)-integrin correlates withthe expression level of native collagenase MMP-1 [48].Therefore, the investigation of this signalling pathwaymight be a promising target for future investigations inthe carcinogenesis of Barrett’s esophagus. However, todate there are no experimental data that support thishypothesis [11].Additionally, the findings of elevated gene and protein

expression of MMP-1 by BE and EAC as preinvasivefactor might also be important for esophageal squa-mous-cell carcinomas, although it was only investigatedin a smaller sample. A critical role of MMP-1 for pro-moting invasion and metastasis in this tumor entity hasbeen described earlier [49].

ConclusionsMMP-1-expression was found in a major population ofproliferating MMP-1+ Barrett and EAC cells. Expressionof MMP-1 in proliferating (Ki-67+) cells of intestinalmetaplasia and in Barrett-associated adenocarcinomasmay thus sustain multi-step carcinogenesis and furthertumor growth. These findings may contribute to thefurther understanding of the pathogenesis of esophagealcarcinogenesis. Furthermore, MMP-1 seems to be in

Table 3 Univariate analysis of prognostic factors of the patients (n = 60)

Variable Unfavorable factor Hazard ratio (HR) 95% CI of HR p-value

LN positive 12.1940 5.9509 to 24.9867 <0.0001

Depth of invasion pT3/4 3.8447 1.5309 to 9.6553 <0.0001

Grading High (G3/4) 4.0652 1.7123 to 9.6514 <0.0001

MMP-1 expression High (≥46%) 1.4526 0.7101 to 2.9718 0.3070

LN, Lymph nodes metastasis.

Figure 4 Overall survival curves calculated by Kaplan-Meiermethod in Barrett associated adenocarcinomas. High levels ofMMP-1 expression in EAC (n = 60) were not found to be associatedwith poorer survival (p = 0.307). The times of the censored data areindicated by short vertical lines.


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respect of the integrin collagen receptor alpha(2)beta(1)-pathway, which has been associated with a putative stemcells hypothesis in prostate cancer. This signaling path-way might be promising for further investigations in thecarcinogenesis of Barrett-associated adenocarcinomasdue to a cancer stem cell hypothesis.

AcknowledgementsThe authors thank the assistance of Mrs. Manuela Schneider and Mrs. SabineGahn for their technical support. We thank the Senator Kurt and IngeSchuster Stiftung, Wuerzburg and the excellence academy of the chairmenof the Deutsche Gesellschaft für Allgemein- und Visceralchirurgie (DGAV) fortheir financial support. For S.G and S.K the work was supported by theWilhelm-Sander Foundation (Grant 2007.068.1).

Author details1Department of General-, Visceral-, Vascular and Pediatric Surgery, Universityof Wuerzburg Hospital, Oberduerrbacher Strasse 6, 97080 Wuerzburg,Germany. 2Department of Cardiac and Thoracic Surgery, University ofWuerzburg Hospital, Oberduerrbacher Strasse 6, 97080 Wuerzburg, Germany.3Institute of Pathology, University of Wuerzburg, Josef-Schneider Strasse 2,97080 Wuerzburg, Germany. 4Institute of Pathology, Medical University ofGraz, Auenbruggerplatz 25, 8036 Graz, Austria.

Authors’ contributionsGM conceived the study, carried out immunohistochemistry studies,performed the statistical analyzes and drafted the manuscript. LMparticipated in the design of the study and performed RT-PCR studies. SKand SG participated in the design of the study, evaluated cancer samples,and helped to draft the manuscript. CR and LS participated in the design ofthe study, and performed immunohistochemistry studies. AH helped to draftthe manuscript. CO and GCT participated in the design of the study designand coordination and drafted the manuscript. VRBHA participated in thedesign of the study design, performed preliminary RT-PCR andimmunohistochemistry studies and drafted the manuscript. All authors readand approved the final manuscript.

Competing interestsThe authors declare that they have no competing interests.

Received: 21 June 2010 Accepted: 14 October 2010Published: 14 October 2010

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Table 4 Multivariate analysis of prognostic factors of the patients (n = 60)

Variable Unfavorable factor Hazard ratio (HR) 95% CI of HR p-value

LN positive 9.1861 2.0665 to 40.8346 0.003746

Depth of invasion pT3/4 1.2336 0.2783 to 5.4683 0.7834

Grading High (G3/4) 2.2593 1.0171 to 5.0186 0.04643

LN, Lymph nodes metastasis.


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doi:10.1186/1479-5876-8-99Cite this article as: Grimm et al.: MMP-1 is a (pre-)invasive factor inBarrett-associated esophageal adenocarcinomas and is associated withpositive lymph node status. Journal of Translational Medicine 2010 8:99.

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